Auxin Responsiveness
Auxins are a group of chemicals which act as plant growth regulators. The class includes, for example, natural compounds such as indole-3-acetic acid (IAA), as well as synthetic auxins such as dicamba, clopyralid, 2,4-dichlorophenoxyacetic acid (2,4-D), and 2,4,5-trichloroacetic acid (2,4,5-T). Auxins are implicated in the regulation of cell extension, cell division, tropisms, vascular differentiation, apical dominance, and root formation. Most, if not all, auxin-induced growth and developmental responses involve alterations in gene expression (Guilfoyle, T. J. (1986) CRC Critical Reviews in Plant Science 4:247-276). Auxin enhances the abundance of a select and conserved set of mRNAs in various plant species, allowing study of gene expression through monitoring of mRNAs, in vitro translation, and cDNA cloning.
“Early genes”, selectively induced in a primary response to auxin prior to the initiation of cell growth, are likely candidates to play a pivotal role in mediating growth-stimulating effects of the hormone. These primary-response genes are induced independent of de novo protein synthesis, indicating direct gene activation. Products of these primary-response genes serve three main functions: emergency rescue and stress adaptation; intercellular communication; and transcriptional regulation of secondary genes to establish and coordinate long-term biological consequences through cascades of auxin signaling. (Abel and Theologis (1996) Plant Physiol. 111:9-17) Thus, primary response genes are of interest both in terms of their activation in response to auxin and in terms of their gene products' downstream effects on plant growth and development.
Multi-gene families of early-auxin-responsive genes have been identified across species. The large Aux/IAA gene family includes related genes from soybean, pea, mung bean, and Arabidopsis. Each is characterized by two to four introns at conserved positions and encodes a small hydrophilic polypeptide with a molecular mass of 19 to 36 kD. (Oeller, P. W., et al. (1993) Journal of Molecular Biology 233:789-798) Aux/IAA mRNAs are specifically induced by biologically active auxins and do not respond to biologically inactive auxin analogs, other plant hormones, or environmental stress. (Guilfoyle, T. J., “Auxin-Regulated Genes and Promoters,” in: Hooykaas, P. J. J., et al., Biochemistry and Molecular Biology of Plant Hormones (Amsterdam, Elsevier, 1999) pp. 423-453)
In contrast, the SAUR (Small Auxin Up RNA) gene family has been studied in a limited number of species. In soybean, five SAUR genes are closely clustered, do not contain introns, and encode highly similar polypeptides of 9 to 10 kD. (Abel and Theologis, supra; Guilfoyle, T. J., 1999, supra) An auxin-induced maize gene involved in coleoptile cell elongation was found to have homology to genes in the SAUR family (Knauss, T., et al., abstract, International Symposium on Auxins and Cytokinins in Plant Development, 26-30 July 1999, Prague, Czech Republic).
Still others types of auxin-responsive genes, such as ACC (1-amino-cyclopropane-1-carboxylic acid) Synthase, involved in ethylene synthesis, may be secondary response genes; i.e., expression is the result of secondary or indirect auxin effects. Several dozen other auxin-responsive cDNA clones from a range of species are less well characterized but demonstrate up- or down-regulation in response to auxin. Expression may also be tissue-specific. (See Tables 1 and 3 in Guilfoyle, T. J., 1999, supra)
A need exists for further characterization of plant responses to auxin. “The list of auxin-induced transcripts continues to grow, emphasizing the plethora of mRNAs that are induced either directly or indirectly by auxin. These mRNAs encode a number of proteins that play potential roles in auxin action and auxin-stimulated growth responses; however, none of these roles in auxin responses has been firmly established.” (Guilfoyle, T. J., 1999, supra, p. 15) Indeed, “despite its critical role in plant development and the immense volume of studies on the diverse auxin effects, understanding of the molecular mechanisms of auxin action remains one of the major challenges of plant biology.” (Abel and Theologis, supra)
Recent studies, including functional tests, have identified putative auxin response elements (AuxREs), conserved sequences in the promoter regions of auxin-responsive genes. Two relatively well-characterized AuxREs are the ocs/as-1 element and the TGTCNC element.
The ocs/as-1 AuxRE was originally identified as an enhancer element in the promoter of the Agrobacterium tumefaciens octopine synthase gene (Ellis et al. (1987) EMBO Journal 6:3203-3208), and similar sequences were subsequently found in the promoter regions of several plant DNA viruses and of soybean and tobacco glutathione S-transferase (GST) genes. It is noted that GST genes may respond not only to exogenous auxins but also to a variety of other hormones, chemical agents, pathogens, and wounding. (Guilfoyle and Hagen, “Potential Use of Hormone-responsive Elements to Control Gene Expression in Plants,” in Inducible Gene Expression (CAB International, 1999)) The element is a 20-bp DNA sequence that consists of an 8-bp direct repeat separated by 4 bp; it has been shown to respond to both biologically active auxins (e.g., IAA, alpha-NAA, 2,4-D, 2,4,5-T) and biologically inactive or weak auxin analogs (e.g., 2,3-D, 2,5-D, 2,6-D, 3,4-D, 3,5-D, 2,4,6-T, and beta-NAA). (see Guilfoyle and Hagen, supra)
In contrast, in the soybean GH3 gene, a composite of two adjacent or overlapping elements, a constitutive element and a TGTCTC element, function in combination to confer responsiveness to biologically active auxins only. (Guilfoyle & Hagen, supra, p. 223) (Guilfoyle, supra, p. 28) Variation in the combination can result in temporal, tissue, and/or developmental specificity of hormone-induced expression for a particular gene. Synthetic composite AuxREs further indicate that the TGTCTC element might function as a global AuxRE within plant genomes and could be coupled with a variety of constitutive elements.
Further identification and characterization of auxin-responsive elements in crop species would be useful in refining control of plant growth and development.
Promoters
Expression of heterologous DNA sequences in a plant host is dependent upon the presence of an operably-linked promoter that is functional within the plant host. Choice of the promoter sequence will determine when and where within the organism the heterologous DNA sequence is expressed.
Where continuous expression is desired throughout the cells of a plant, constitutive promoters are utilized. Examples of constitutive promoters include the cauliflower mosaic virus (CaMV) 35S transcription initiation region, the Nos, 1′- or 2′-promoter derived from T-DNA of Agrobacterium tumefaciens, the ubiquitin 1 promoter, the Smas promoter, the cinnamyl alcohol dehydrogenase promoter (U.S. Pat. No. 5,683,439), the pEmu promoter, the rubisco promoter, the GRP1-8 promoter, and other transcription initiation regions from various plant genes known to those of skill.
Alternatively, the plant promoter can direct expression of a polynucleotide of the present invention in a specific tissue or may be otherwise under more precise environmental or developmental control. Where gene expression in response to a stimulus is desired, inducible promoters are the regulatory element of choice. The ability to selectively induce the expression of specific genes allows for the manipulation of development and function not only at the cellular level, but also at the system and organismal level. Generally, a specific nucleotide sequence known as a response element is located in the 5′ regulatory region of a target gene that is activated by the stimulus. Environmental conditions that may effect transcription by inducible promoters include pathogen attack, anaerobic conditions, or the presence of light. Examples of inducible promoters are the Adhl promoter which is inducible by hypoxia or cold stress, the Hsp70 promoter which is inducible by heat stress, and the PPDK promoter which is inducible by light.
Where expression in specific tissues or organs is desired, tissue-preferred promoters are used; these promoters can preferentially drive expression in specific tissues or organs. Examples of promoters under developmental control include promoters that initiate transcription only, or preferentially, in certain tissues, such as leaves, roots, fruit, seeds, or flowers. Exemplary promoters include the anther-specific promoter 5126 (U.S. Pat. Nos. 5,689,049 and 5,689,051), glb-1 promoter, and gamma-zein promoter. See U.S. Pat. No. 5,986,174 for a discussion of methods to identify tissue-preferred transcriptional regulatory elements.
Additional regulatory sequences upstream and/or downstream from the core promoter sequence can be included in expression cassettes of transformation vectors to bring about varying levels of expression of heterologous nucleotide sequences in a transgenic plant.
Reproductive Biology
Control of pollen fertility is essential in hybrid crop production. In hybrid maize production, control of pollen fertility is typically accomplished by physically removing the male inflorescence, or tassel, prior to pollen shed. Manual detasseling is highly labor-intensive. Although mechanical detasseling is less labor-intensive than manual detasseling, mechanical detasseling is less reliable; it requires subsequent examination of the crop and may require remedial manual detasseling. Both methods of detasseling cause a reduction of yield.
Pollen fertility can also be controlled by applying a chemical composition to the plant or soil to prevent pollen production in female plants. See, for example, Ackrnann et al., U.S. Pat. No. 4,801,326. According to this method, hybrid seeds are produced by the fertilization of the treated female plants with pollen from non-treated plants. However, the chemical approach is labor-intensive and presents potential problems with the toxicity of chemicals introduced into the environment.
Another approach to the control of fertility is based upon the use of a cytoplasmic gene(s) for male sterility. See, for example, Patterson, U.S. Pat. No. 3,861,079. The problem with this approach, however, is that the expression of certain cytoplasmic male sterility genes is accompanied by increased susceptibility to fungal pathogens. For example, extensive use of one cytotype, cmsT, led to an epiphytic outbreak of Southern Corn Leaf Blight in the early 1970's. Although additional cms cytotypes have become available, their use has not become widespread due to the concern over possible susceptibility to the Southern Corn Leaf Blight pathogen, or to other, as yet unknown, pathogens.
One type of genetic sterility is disclosed in U.S. Pat. Nos. 4,654,465 and 4,727,219 to Brar, et al. However, this form of genetic male sterility requires maintenance of multiple mutant genes at separate locations within the genome and requires a complex marker system.
Other attempts have been made to improve on these methods to control fertility. For example, see EPO 0329308 and WO 90/08828, which describe an antisense system in which a gene critical to fertility is identified and an antisense construct to that gene is used to generate sterility. U.S. Pat. No. 5,426,041 describes a method of transforming plants to produce a gene product which interferes with pollen formation and/or function.
In summary, a functional promoter which can be induced by exogenous application of an auxin and which results in preferential expression in specific tissues or organs is of interest. A need continues to exist for novel methods of controlling fertility in maize plants. Combination of the inducible, tissue-preferred promoter with a gene to overcome male sterility would be useful, particularly in hybridization of maize.
Transformation
Current methods for genetic engineering in maize require a specific cell type as the recipient of new DNA. These cells are found in relatively undifferentiated, rapidly-growing callus cells, or on the scutellar surface of the immature embryo (which gives rise to callus). Irrespective of the delivery method currently used, DNA is introduced into thousands of cells, yet stably-transformed cells are recovered at frequencies of 10−5 relative to transiently-expressing cells. Exacerbating this problem, the trauma that accompanies DNA introduction directs recipient cells into cell cycle arrest, and evidence suggests that many of these cells are directed into apoptosis or programmed cell death. (Bowen et al., Third International Congress of the International Society for Plant Molecular Biology, 1991, Abstract 1093). Therefore it would be desirable to provide improved methods capable of increasing transformation efficiency.
While significant advances in plant transformation have been made over the last few years, in major crop plants, such as maize and soybeans, serious genotype limitations still exist. Transformation of model genotypes is efficient, but many elite genotypes fail to produce a favorable culture response, and the process of introgressing transgenes into production inbreds is laborious, expensive and time-consuming. One approach to improving recovery of transformants from culture is through expression of polynucleotides which may enhance tissue culture response, induce somatic embryogenesis, induce apomixis, increase transformation efficiency and/or increase recovery of regenerated plants. This would include, for example, expression in transformed cells of a LEC1 (leafy cotyledon 1) polynucleotide (U.S. patent application Ser. No. 09/435,054 and WO 00/28058, hereby incorporated by reference). However, a preferred promoter is needed to optimize improvements in transformation efficiency such as that conferred by LEC1.